ABSTRACT Protein acetylation is the most ancient and common form of posttranslational modification, and the vast majority of the human proteome is acetylated. Protein acetylation is mediated by protein lysine acetyltransferases (KATs), which are grouped into Histone ATs (HATs) and non-histone ATs, and protein N-terminal ATs (NATs). In mammalian cells, KATs acetylate thousands of proteins, spanning a wide class spectrum, including transcription factors, kinases, ubiquitinligases, ribosomal proteins and metabolic enzymes, and mediating a broad range of cellular activities, including cell cycle control, DNA damage check-points, cytoskeleton organization, endocytosis and metabolism. The posttranslational and cotranslational process of N-terminal acetylation by NATs occurs on ~85% of human proteins and is also involved in numerous biological processes including cellular apoptosis, enzyme regulation, protein localization, rDNA transcriptional regulation and protein degradation. Aberrant AT activities have also been associated with several diseases including solid and haematological cancers, rare genetic disorders, and metabolic and neurodegenerative disorders, thus implicating ATs as attractive drug targets for therapy. Despite the importance of ATs, mechanistic information is largely limited to the isolated catalytic AT domains, and the critical role played by AT cofactor and auxiliary proteins in mediating AT-regulated cellular pathways are largely unknown. In addition, potent, selective and cell permeable AT inhibitors as molecular probes for AT-mediated pathways and as lead molecules for therapy are generally not available. The overall goal of this proposal is to understand the molecular mechanisms of protein acetylation by HATs, non-histone KATs and NATs, with a particular focus on addressing the following unresolved and important questions and goals in the field: (A) How are HATs regulated by cofactor proteins for substrate-specific acetylation? (B) What are the unique AT properties of non-histone KATs? (C) How do auxiliary proteins and ribosome association contribute to NAT function? (D) Can we leverage mechanistic and structural information to develop potent and selective protein AT inhibitors?